1. Field of the Invention
This invention is directed to capillary electrophoresis detection, in general, and to precision detector assemblies using optical detection, in particular.
2. Prior Art
Capillary electrophoresis is an important emerging new technology. It offers significant advantages in separation of a wide range of compounds ranging from small ions to hole cells. The separation can be accomplished in several ways. The solution can be supplied to a medium of paper, gel starch or a liquid carrier buffer. The preferred method of interest herein is the latter method. That is, a solution is prepared which comprises a carrier buffer and a sample to be evaluated. This solution is stored in an input reservoir and, selectively, transported to an output reservoir. Typically, the solution is transported through a tube which is transparent (or at least translucent). The solution can be caused to be transported through the tube by applying a pressure or an electrical differential between the respective reservoirs. As the solution passes through the tube, it tends to separate. This is a well-known technique in the art.
One of the difficulties associated with the technology lies in the detection of the separated components as they migrate along the capillary column.
In the past, a technique reported by Tiselius (circa 1929) utilized an open U-tube filled with a buffer liquid and the sample. (This is sometimes referred to as the Schlieren technique.) In this arrangement, the separation of the sample components in the U-tube was visually observed. Obviously, this technique was extremely crude and provided relatively low accuracy results. For example, the tube was relatively large in diameter which did not cause the sample to separate to a fine degree. Also, the optics involved provided relatively gross observations and little detailed analysis.
Later (circa 1974), in a technique reported by Virtannen, separation of the samples in the observation tube was enhanced. This technique, referred to as isotachophoresis, improved the sample separation, but the optical observation apparatus was not significantly improved.
In or around 1980, Hjerten and Jorgenson developed the small bore tube (or capillary) technique for separation. This capillary approach was a significant development in electrophoresis technique. It provided several advantages including a minimizing effect of diffusion, a high number of theoretical plates and a smaller diameter observation field.
In this technique, light was directed across the capillary to enhance viewing of the sample. However, in this arrangement the light source and receiver were disposed outside the capillary tube. Moreover, the light source and receiver were, typically, disposed outside of a support sleeve in which the capillary was mounted. Thus, significant optical problems existed. For example, the perturbations and wall effects produced by the sleeve and/or capillary created some distortions and/or anomalies in the observed data. Because the sleeve and/or capillary presented cylindrical surfaces and because of the differences in indices of refraction, some of the incident light rays completely missed the sample. Likewise, some of the incident light rays traversed paths which were less than the entire sample. While these effects can be calculated to some degree and factored into the resultant data, this information is less than perfect and subject to question.
In 1987, Karger developed the gel column which has a significant sieving effect on the sample. However, the optics in this system are not significantly better than the other prior art devices.
The physical properties of the optics in such systems is important as the absorbance detection of samples is dependent upon the molar extinction (at a known wavelength) of the absorbing material, the concentration of the specimen and the path length through which the radiation must pass.
In other words, the equivalent absorbance A in this sample measuring technique is defined as EQU A=kcl
where
k=the molar extinction coefficient;
c=concentration; and
1=path length through solution.
In a given analysis situation, the only component of this equation which can be effectively controlled is 1. This factor is controlled by the optics of the apparatus. Thus, an improved optical arrangement is highly desirable in the electrophoresis measuring technology.
One of the problems in optical detection systems is stray light which, in general, may be described as any energy which reaches the detector without having been affected by the sample. Stray light can arise from either the direct optical path or from an external path.
The small size of the capillary column demands accuracy in placement of an optical detection system since most optical systems presently employed are located outside the capillary.
With the capillaries of vanishingly small dimensions (5-200 microns), extreme precision is required to assure that interrogating radiation passes through the bore of the capillary where the sample to be detected is located and, as well, to avoid any radiation "piping" around the bore through the capillary material or even passing by the capillary totally.
With optical fibers, there is an angle of acceptance which contains and propagates the light. A numerical aperture, which is the sine of half the angle of acceptance for the fiber, defines the angle of propagation through the fiber. Light entering the fiber at angles greater than this value leaks away and is not propagated to the output end.
The present invention provides an apparatus with the optical detection system inside the capillary column, thereby eliminating alignment difficulties, and other advantages disclosed herein.